Isolation and identification of arctiin and arctigenin in leaves of burdock (Arctium lappa L.) by polyamide column chromatography in combination with HPLC-ESI/MS

A simple method involving polyamide column chromatography in combination with HPLC-PAD and HPLC-ESI/MS for isolating and identifying two kinds of lignans, arctiin and arctigenin, in the leaves of burdock (Arctium lappa L.) has been established. After extraction of burdock leaves with 80% methanol, the aqueous phase of crude extracts was partitioned between water and chloroform and the aqueous phase was fractionated on a polyamide glass column. The fraction, eluting with 100% methanol, was concentrated and gave a white precipitate at 4 degrees C from which two main compounds were purified by semi-preparative HPLC. In comparison with the UV and ESI-MS spectra and the HPLC retention time of authentic standards, the compounds were determined to be arctiin and arctigenin. The extraction/separation technique was validated using an internal standard method.     

Determination of the major constituents in fruit of Arctium lappa L. by matrix solid-phase dispersion extraction coupled with HPLC separation and fluorescence detection

The arctiin and arctigenin in the fruit of Arctium lappa L. were extracted by matrix solid-phase dispersion (MSPD) and determined by high-performance liquid chromatography (HPLC) with fluorescence detection. The experimental conditions for the MSPD were optimized. Silica gel was selected as dispersion adsorbent and methanol as elution solvent. The calibration curve showed good relationship (r > 0.9998) in the concentration range of 0.010–5.0 μg mL⁻¹ for arctiin and 0.025–7.5 μg mL⁻¹ for arctigenin. The recoveries were between 74.4% and 100%. The proposed method consumed less sample, time and solvent compared with conventional methods, including ultrasonic and Soxhlet extraction.     

Americanin, a bioactive dibenzylbutyrolactone lignan, from the seeds of Centaurea americana

The reversed-phase preparative HPLC analysis of the methanol (MeOH) extract of the seeds of Centaurea americana afforded a dibenzylbutyrolactone lignan, 3'-O-caffeoyl arctiin (named americanin), together with five known lignans, arctiin, arctigenin, matairesinol, matairesinoside and lappaol A, and two known phytoecdysteroids, 20-hydroxyecdysone and makisterone A. While the structures of the known compounds were determined by direct comparison of the spectral data with published data, the structure of americanin was elucidated by UV, MS and a combination of 1D and 2D NMR spectral analyses. The antioxidant properties and toxicity of the extracts and the isolated compounds were determined by the DPPH and the brine shrimp lethality assays, respectively.     

Analysis of Binding Interaction Between Antibacterial Ciprofloxacin and Human Serum Albumin by Spectroscopic Techniques

To develop an efficient method for extracting and purifying the active ingredient, arctiin, from Fructus arctii and to investigate the protective effect of arctiin against glucose-induced rat aortic endothelial cell (RAEC) injury was investigated. Using a L9 (34) orthogonal array and two-step column chromatography (with AB-8 macroporous resin) arctiin extraction was optimized using a reflux method with 70 % ethanol. The RAECs were then treated with different concentrations of arctiin (1, 10, or 100 μg/ml). The effects of arctiin on cell viability in a high glucose medium, malondialdehyde (MDA) levels, and lactate dehydrogenase were measured using commercially available assays. After extraction, the purity of arctiin reached 95.7 %. In rats, arctiin was shown to stimulate the proliferation of RAECs in a high glucose medium in a dose-dependent manner. Exposure of RAECs to high glucose resulted in a significant increase in MDA and release of lactate dehydrogenase. This was accompanied by significant increase in nitric oxide release and expression of antiendothelial nitric oxide synthase. This technique resulted in relatively pure arctiin extraction. Furthermore, the results from this study suggest that arctiin could potentially function as a protector against vascular endothelial cell injury and further investigation is warranted.     

Arctigenin,a Natural Lignan Compound,Induces Apoptotic Death of Hepatocellular Carcinoma Cells via Suppression of PI3‐K/Akt Signaling

In this study, we explored the cytotoxic effects of arctigenin, a natural lignan compound, on human hepatocellular carcinoma (HCC) cells and check the involvement of phosphatidylinositol 3‐kinase (PI3‐K)/Akt signaling. HCC cells were treated with different concentrations of arctigenin and cell viability and apoptosis were assessed. Manipulating Akt signaling was used to determine its role in the action of arctigenin. Arctigenin significantly inhibited the viability of HCC cells in a concentration‐dependent manner. Arctigenin induced apoptosis and activation of caspase‐9 and ‐3. Overexpression of a constitutively active Akt mutant blocked arctigenin‐induced apoptosis. Combinational treatment with arctigenin and the PI3‐K inhibitor LY294002 significantly enhanced apoptosis. Arctigenin reduced the expression of Bcl‐xL, Mcl‐1, and survivin and the phosphorylation of mTOR and S6K, which were significantly reversed by overexpression of constitutively active Akt. This is the first report about the anticancer activity of arctigenin in HCC cells, which is mediated by inactivation of PI3‐K/Akt signaling.     

Arctigenin Exhibits Relaxation Effect on Bronchus by Affecting Transmembrane Flow of Calcium

Arctigenin, a lignan extract from Arctium lappa (L.), exhibits anti-inflammation, antioxidation, vasodilator effects, etc. However, the effects of arctigenin on bronchus relaxation are not well investigated. This study aimed to investigate how arctigenin regulates bronchus tone and calcium ion (Ca²⁺) flow. Trachea strips of guinea pigs were prepared for testing the relaxation effect of arctigenin to acetylcholine, histamine, KCl, and CaCl₂, respectively. Furthermore, l-type calcium channel currents were detected by patch–clamp, and intracellular Ca²⁺ concentration was detected by confocal microscopy. The results showed that arctigenin exhibited relaxation effect on tracheae to different constrictors, and this was related to decreasing cytoplasmic Ca²⁺ concentration by inhibiting Ca²⁺ influx partly through l-type calcium channel as well as promoting Ca²⁺ efflux. In summary, this study provides new insight into the mechanisms by which arctigenin exhibits relaxation effect on bronchus and suggests its potential use for airway disease therapy.     

Simultaneous HPLC analysis, with isocratic elution, of glycyrrhizin and glycyrrhetic acid in liquorice roots and confectionery products

Glycyrrhizin (1), the main active principle of Glycyrrhiza glabra (liquorice) roots, is extensively used in herbal medicines, in pharmaceutical preparations and confectionery products. A feasible and reliable method which allows the simultaneous analysis of 1 and its aglycone, 18beta-glycyrrhetic acid (2), by means of an isocratic HPLC procedure is described. The system uses a C8 column as the stationary phase, and a mixture of acetonitrile, methanol, water and glacial acetic acid as the mobile phase. Good linearity was found in the concentration ranges 1-50 and 0.05-2.50 microg/mL for 1 and 2, respectively. A simple and rapid sample pre-treatment, based on the extraction of the two analytes with a mixture of water and ethanol, was developed for the examination of liquorice confectionery products and root samples. The HPLC method was shown to be appropriate, in terms of precision and feasibility, for the quality control of the analytes in these matrices.     

Natural lignans from Arctium lappa modulate P-glycoprotein efflux function in multidrug resistant cancer cells

Arctium lappa is a well-known traditional medicinal plant in China (TCM) and Europe that has been used for thousands of years to treat arthritis, baldness or cancer. The plant produces lignans as secondary metabolites which have a wide range of bioactivities. Yet, their ability to reverse multidrug resistance (MDR) in cancer cells has not been explored. In this study, we isolated six lignans from A. lappa seeds, namely arctigenin, matairesinol, arctiin, (iso)lappaol A, lappaol C, and lappaol F. The MDR reversal potential of the isolated lignans and the underlying mechanism of action were studied using two MDR cancer cell lines, CaCo2 and CEM/ADR 5000 which overexpress P-gp and other ABC transporters. In two-drug combinations of lignans with the cytotoxic doxorubicin, all lignans exhibited synergistic effects in CaCo2 cells and matairesinol, arctiin, lappaol C and lappaol F display synergistic activity in CEM/ADR 5000 cells. Additionally, in three-drug combinations of lignans with the saponin digitonin and doxorubicin MDR reversal activity was even stronger enhanced. The lignans can increase the retention of the P-gp substrate rhodamine 123 in CEM/ADR 5000 cells, indicating that lignans can inhibit the activity of P-gp. Our study provides a first insight into the potential chemosensitizing activity of a series of natural lignans, which might be candidates for developing novel adjuvant anticancer agents.     

36种中药材体外抗菌活性筛选研究

该文研究36种常用中药材80%乙醇提取物在体外抗临床常见致病菌的抗菌活性。采用药敏纸片法测耐药菌的耐药谱,中药粗粉用80%乙醇浸泡提取,提取液减压浓缩得浸膏,通过琼脂打孔法测定提取物抑菌圈,再通过微量倍比稀释法测定最低抑菌浓度(MIC)和最低杀菌浓度(MBC)。结果表明:36种中药材醇提物中,有15种具有广谱抗菌活性,对实验中各标准菌表现出不同程度的抑制作用,对MRSA抗菌活性也较强。其中,岩陀、卷柏、首乌藤、苏木、乌药、夏枯草6种药材的抗菌活性比较突出,抑菌圈均大于11 mm,细菌对其表现为中高度敏感;它们对7株标准菌的MIC/MBC值除个别为12.5 mg·m L~(-1)以外,均小于1.563 mg·m L~(-1),对16株MRSA的MIC/MBC值均小于1.563 mg·m L~(-1),它们的萃取层活性均小于1 mg·m L~(-1)。所筛选出的15种抗菌活性较强的中药材,可为后续研究其活性单体化合物和作用机制,研发有效的抗多重耐药菌的中药制剂以及解决细菌耐药性问题提供一定的参考。     

A HPLC method for the analysis of germacrone in rabbit plasma and its application to a pharmacokinetic study of germacrone after administration of zedoary turmeric oil

A validated new, simple and highly sensitive reversed-phase HPLC method is developed for studying the pharmacokinetics of germacrone after intravenous administration of zedoary turmeric oil (ZTO) oil-in-water microemulsion. The method did not require a complex and expensive equipment. A high extraction recovery (>80%) of germacrone was obtained. Linear calibration curves obtained with the peak-area ratio (y) of germacrone to internal standard (tanshinoneIIA) versus drug concentration (x) were found to be linear between 8.08 and 808 ng/ml. The limit of quantitation was 8.08 ng/ml.The monitored compounds were completely separated from others in ZTO and from endogenous species in plasma by HPLC. Pharmacokinetic investigations were performed on 18 male rabbits after intravenous administration of ZTO microemulsion via the ear vein at germacrone doses of 3.2, 6.4 and 9.6 mg/kg. The plasma concentration-time data fit to a two-compartment intravenous model with a weight of 1/C(2) (C: germacrone concentration in plasma). Germacrone exhibited linear pharmacokinetics after intravenous administration of ZTO microemulsion to rabbits over the germacrone dose range 3.2-9.6 mg/ml.     

Design and development of microemulsion drug delivery system of acyclovir for improvement of oral bioavailability

The main purpose of this work was to develop an oral microemulsion formulation for enhancing the bioavailability of acyclovir. A Labrafac-based microemulsion formulation with Labrasol as surfactant and Plurol Oleique as cosurfactant was developed for oral delivery of acyclovir. Phase behavior and solubilization capacity of the microemulsion system were characterized, and in vivo oral absorption of acyclovir from the microemulsion was investigated in rats. A single isotropic region, which was considered to be a bicontinuous microemulsion, was found in the pseudoternary phase diagrams developed at various Labrasol:Plurol Oleique:Labrafac ratios. With the increase of Labrasol concentration, the microemulsion region area and the amount of water and Labrafac solubilized into the microemulsion system increased; however, the increase of Plurol Oleique percentage produced opposite effects. The microemulsion system was also investigated in terms of other characteristics, such as interfacial tension, viscosity, pH, refractive index, diffusion, and bioavailability. Acyclovir, a poorly soluble drug, displayed high solubility in a microemulsion formulation using Labrafac (10%). Labrasol (32%), Plurol Oleique (8%), and water (50%). The in vitro intraduodenal diffusion and in vivo study revealed an increase of bioavailability (12.78 times) after oral administration of the microemulsion formulation as compared with the commercially available tablets. Published: September 15, 2006     

Steroid bioconversion in a microemulsion system

The Delta(1,2)-dehydrogenation of high concentrations of the steroid -methyl-Reichstein's compound S-21-acetate (16MRSA) in a microemulsion system was studied using heat-dried and thawed Arthrobacter simplex cells as biocatalyst. The microemulsion system consists of an organic phase [75-95% (v/v)] with steroid (1-60 g/L(tot)), an aqueous phase [5-25% (v/v)] containing the cells (5-30 g/L(tot)), and a neutral surfactant (5-20 g/L organic solvent). Benzene derivatives, which solubilize 16MRSA up to 94 g/L, and phospholipids were used as organic solvents and surfactants, respectively, and menadione was added as an external electron acceptor. Factors affecting the dehydrogenation rate in the microemulsion system were studied. The influences of the 16MRSA and the menadione concentration on the dehydrogenation rate were described by Michaelis-Menten kinetics, apparent V'(max) and K'(m) values of 2.06 g/g dry weight h and 18.9 g/L for 16MRSA and 4.97 g/g dry weight h and 1.91 g/L for menadione being obtained. Optimal menadione concentration was dependent on the steroid concentration was dependent on the steroid concentration used. The reaction was strongly inhibited by high product concentrations. Much higher activities were obtained with the thawed cells than with the dried cells, conversions of 98% being reached within 14-16 h. for 16MRSA and cell dry weight concentrations of 40 and 10 g/L, respectively. Activity retention in a batch stirred tank reactor remained constant during the first 16-24 h of operation and then decreased, depending on the stirring rate; 22 to 65% of the initial reaction rate was obtained after 48 h at stirring rates of 650 and 2000 rpm, respectively.     

Squalene epoxide cyclase and microemulsion

Squalene epoxide cyclase was extracted from microsomal preparations of rat liver using anionic, cationic and non-ionic microemulsions. The anionic microemulsion was the best with respect to protein solubilisation, extracted cyclase activity and stability of this activity over time. The activity assay showed cyclase activity to be higher in anionic microemulsion than in buffer in the presence of surfactant. Calcium chloride in the anionic microemulsion had a stabilising effect and less total protein seemed to be extracted.     

Stability and activity of alcohol dehydrogenases in W/O-microemulsions: enantioselective reduction including cofactor regeneration

Microemulsions provide an interesting alternative to classical methods for the conversion of less water-soluble substrates by alcohol dehydrogenase, but until now stability and activity were too low for economically useful processes. The activity and stability of the enzymes are dependent on the microemulsion composition, mostly the water and the surfactant concentration. Therefore, it is necessary to know the exact phase behavior of a given microemulsion reaction system and the corresponding enzyme behavior therein. Because of their economic and ecologic suitability polyethoxylated fatty alcohols were investigated concerning their phase behavior and their compatibility with enzymes in ternary mixtures. The phase behavior of Marlipal O13-60 (C13EO6 in industrial quality)/cyclohexane/water and its effect on the activity and stability of alcohol dehydrogenase from Yeast (YADH) and horse liver (HLADH) and the carbonyl reductase from Candida parapsilosis (CPCR) is presented in this study. Beside the macroscopic phase behavior of the reaction system, the viscosity of the system indicates structural changes of aggregates in the microemulsion. The changes of the enzyme activities with the composition are discussed on the basis of transitions from reverse micelles to swollen reverse micelles and finally, the transition to the phase separation. The formate dehydrogenase from Candida boidinii was used for the NADH-regeneration during reduction reactions. While the formate dehydrogenase did not show any kinetic effect on the microemulsion composition, the other enzymes show significant changes of activity and stability varying the water or surfactant concentration of the microemulsion. Under certain conditions, stability could be maintained with HLADH for several weeks. Successful experiments with semi-batch processes including cofactor regeneration and product separation were performed.     

Assessment of genotoxicity of herbal medicinal products: Application of the “bracketing and matrixing” concept using the example of Valerianae radix (valerian root)

An assessment of genotoxicity is a precondition for marketing authorization respectively registration of herbal medicinal products (HMPs), as well as for inclusion into the ‘Community list of herbal substances, preparations and combinations thereof for use in traditional herbal medicinal products’ established by the European Commission in accordance with Directive 2001/83/EC as amended, and based on proposals from the Committee on Herbal Medicinal Products (HMPC).In the ‘Guideline on the assessment of genotoxicity of herbal substances/preparations’ (EMEA/HMPC/107079/2007) HMPC has described a stepwise approach for genotoxicity testing, according to which the Ames test is a sufficient base for the assessment of genotoxicity in case of an unequivocally negative result. For reducing efforts for testing of individual herbal substances/preparations, HMPC has also developed the ‘guideline on selection of test materials for genotoxicity testing for traditional herbal medicinal products/herbal medicinal products’ (EMEA/HMPC/67644/2009) with the aim to allow testing of a standard range of test materials which could be considered representative of the commonly used preparations from a specific herbal drug according to a ‘bracketing/matrixing’ approach.The purpose of this paper is to provide data on the practical application of this bracketing and matrixing concept using the example of Valerianae radix, with the intention of facilitating its inclusion in the “Community list”. Five extraction solvents, representing the extremes of the polarity range and including also mid-range extraction solvents, were used, covering the entire spectrum of phytochemical constituents of Valerianae radix, thereby including polar and non-polar constituents. Extracts were tested in the Ames test according to all relevant guidelines. Results were unequivocally negative for all extracts. A review of the literature showed that this result is in accordance with the available data, thus demonstrating the lack of a genotoxic potential.In conclusion the two guidelines on genotoxicity provide a practically applicable concept. Valerianae radix has no genotoxic potential, supporting its use in HMPs and its inclusion in the Community list.     

Determination of three bile acids in artificial Calculus Bovis and its medicinal preparations by micellar electrokinetic capillary electrophoresis

A micellar electrokinetic capillary electrophoresis (MEKCE) method for the determination of cholic acid (CA), hyodeoxycholic acid (HDCA) and chenodeoxycholic acid (CDCA) in artificial Calculus Bovis and its four medicinal preparations is described. The buffer solution consisted of 40 mM disodic phosphate and 40 mM sodium dodecylsulfate (SDS) adjusted to pH 9.0. UV detection was set to 200 nm. Under optimum conditions, the analytes were baseline separated within 11min. The linear calibration range was 12.1-970 microgml(-1) for CA and 18.8-950 microgml(-1) for HDCA and CDCA, respectively. It was found that overall recoveries were within the range of 98-102%, and R.S.D.s were less than 5% for the analytes. This method, due to its convenience, high accuracy and good reproducibility can be employed in quality control of artificial Calculus Bovis and its medicinal preparations.     

IgG4 levels in polyvalent immunoglobulins and immunoglobulins prepared by precipitation with caprylic acid

Levels of IgG4 in immunoglobulin preparations obtained either by conventional ethanol fractionation or by ethanol and caprylic acid fractionation (Allergam) were measured by an enzyme-immunoassay (competitive Elisa). In 9 ethanol preparations, the mean percentage of IgG4 was 2.1% +/- 0.5. In 10 preparations of Allergam, the mean percentage of IgG4 was 4.6% +/- 1. The concentration of IgG4 in Allergam preparations is about twice the concentration found in conventional ethanol preparations.     

Extraction methods and chemical standardization of botanicals and herbal preparations

Botanicals and herbal preparations are medicinal preparations, containing a single or two or more medicinal plants. The focus of this review paper is on the analytical methodologies, which included the combination of sample preparation tools and chromatographic techniques for the chemical standardization of marker compounds or active ingredients in botanicals and herbal preparations. The common problems and key challenges in the chemical standardization of botanicals and herbal preparations were discussed. As sample preparation is the most important step in the development of analytical methods for the analysis of constituents present in botanicals and herbal preparations, the strength and weakness of different extraction techniques are discussed. For the analysis of compounds present in the plant extracts, the applications of common chromatographic techniques, such as HPLC, CE, HRGC/MS, HPLC/MS and HPLC/MS/MS are discussed. The strength, weakness and applicability of various separation tools are stated. Procedures for the identification of marker or active compounds in plant extracts, using HPLC/MS, were proposed. Finally, the effects of batch-to-batch variation of the medicinal plants are investigated and discussed.